Can Heritable Epigenetic Variation Aid Speciation?

Hindawi Publishing Corporation Genetics Research International Volume 2012, Article ID 698421, 9 pages doi:10.1155/2012/698421

Review Article Environmental Heterogeneity and Phenotypic Divergence: Can Heritable Epigenetic Variation Aid Speciation?

Ruth Flatscher,1, 2 Bozoˇ Frajman,1 Peter Schonswetter,¨ 1 and Ovidiu Paun2

1 Institute of Botany, University of Innsbruck, Sternwartestraße 15, 6020 Innsbruck, Austria 2 Department of Systematic and Evolutionary Botany, University of Vienna, Rennweg 14, 1030 Vienna, Austria

Correspondence should be addressed to Ovidiu Paun, [email protected]

Received 22 August 2011; Revised 7 November 2011; Accepted 23 November 2011

Academic Editor: Christina L. Richards

Copyright © 2012 Ruth Flatscher et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The dualism of genetic predisposition and environmental influences, their interactions, and respective roles in shaping the phenotype have been a hot topic in biological sciences for more than two centuries. Heritable epigenetic variation mediates between relatively slowly accumulating mutations in the DNA sequence and ephemeral adaptive responses to stress, thereby providing mechanisms for achieving stable, but potentially rapidly evolving phenotypic diversity as a response to environmental stimuli. This suggests that heritable epigenetic signals can play an important role in evolutionary processes, but so far this hypothesis has not been rigorously tested. A promising new area of research focuses on the interaction between the different molecular levels that produce phenotypic variation in wild, closely-related taxa that lack genome-wide genetic differentiation. By pinpointing specific adaptive traits and investigating the mechanisms responsible for phenotypic differentiation, such study systems could allow profound insights into the role of epigenetics in the evolution and stabilization of phenotypic discontinuities, and could add to our understanding of adaptive strategies to diverse environmental conditions and their dynamics.

1. Introduction of biological variation: the genetic system defines the range of functional possibilities of each individual. However, Patterns and causes of biological variation have fascinated these heritable differences translate into the phenotype and challenged natural scientists for a long time. The only indirectly via the resulting RNA and protein products Darwinian evolutionary theory highlights the importance which mould the structure and function of an organism. of natural variation as raw material upon which selection Much progress has been made in recent years in identifying processes can act, thereby increasing the fitness of locally gene functions and candidate genes coding for important adapted phenotypes [1]. Conceptual and technical develop- metabolic enzymes, but analyses of whole genomes remain a ments since the late 19th century have greatly enhanced our complex challenge. Even in organisms whose whole genome understanding of some of the main mechanisms producing is sequenced, a large number of genes still remain unchar- and maintaining biological variation, namely, genetic muta- acterized [8]. The second important source of biological tion and recombination [2]. However, natural selection acts variation is fluctuation in rates of gene expression, resulting upon phenotypic variation represented by the individual [3], in phenotypic plasticity [9, 10]. Genes can be up- or down- which is delimited by its genetic constitution, but also shaped regulated in response to environmental conditions, such as by its specific environment [4] and developmental processes temperature regimes or water supply, or intrinsic factors [5]. The process of evolution is thus a result of complex such as specific phenological or developmental stages [11]. interactions between various intrinsic and extrinsic factors This leads to temporary modifications of the phenotype, [6]. which are generally not passed on to the next generation [12, Therefore, current evolutionary investigations should 13]. The third level, heritable epigenetic variation, via both consider several levels of biological variation [7]. First, specialized enzymology inducing structural modifications of differences in the DNA sequence account for a great amount the DNA (through DNA methylation, histone acetylation 2 Genetics Research International

[14, 15]) and small interfering (si) RNA populations [16, 17], lacking genetic variability [45] and/or occupying a frag- results in (meta) stable chromatin landscape differences. mented landscape. Selectable epigenetic variation can enable Epigenetic differences determine if and where particular genetically depauperate lineages to adapt [46] until genetic genes or groups of genes are to be expressed, while the under- assimilation occurs (i.e., when environmentally induced lying DNA sequence remains identical [18]. Most of these phenotypic variation becomes fixed by secondary genetic differences are reversible developmental effects and they control, e.g., after deamination of methylated cytosine to are part of the molecular processes underlying phenotypic thymine [13, 47]). Thus, heritable epigenetic variation could plasticity in response to variation in the environment [19]. pave the way for genetic adaptation. However, environmental change, severe stress or genomic The epigenetic sources of variation can be stochastic shock events like hybridization or genome duplication can epimutations, but a major part of the epigenetic variation change the epigenetic configuration of an organism resulting is triggered by stress or changes in the environment [3, in new phenotypes [20–26], and some of these alterations 22, 48], that is, under circumstances when new phenotypes can be passed on to the next generations [27–30]. could be crucial for survival. Moreover, if conditions return The molecular mechanisms underlying these compo- to their original state, spontaneous back-mutation of epi- nents of phenotypic variation differ in their stability and in alleles can restore original phenotypes (e.g., in position- the time frames in which they confer phenotypic novelty. The effect variegation [27]). In the light of epigenetic variation, genetic sequence is the most stable, evolving slowly through the involvement of the environment in evolution becomes mutation and gradually accumulating changes over a large twofold: as a stimulant of variation and as the selector of number of generations. In contrast, gene expression levels adaptive variation. can be rapidly and continuously regulated within a very At the interface between genotype and environment, the short time [11], much shorter than the generation length of overall rate of epimutations is often much higher than that an organism, and allow an almost instantaneous response of genetic mutations [49], resulting in a more dynamic level of the individual to its environment within limits defined of variation. Novel epigenetic modifications may originate by its genetic constitution. Heritable epigenetic alterations simultaneously in several individuals in a population under act within an intermediate time horizon, since they can stress, which will facilitate fixation. Despite the potentially occurasanimmediateandmultilocusreactiontodifferent high loss of epigenetic novelties by epigenetic reset [19], kinds of external or intrinsic stimuli [23] but are not as epimutations can reach equilibrium frequencies within pop- ephemeral as plastic gene regulation and can affect the ulations rapidly, over less than a dozen generations if the following generations [18]. environmental stress is maintained long enough [28]. In It has long been established that mutations in DNA stark contrast to the expected incidence of genetic mutations, sequence are the primary raw material for evolutionary environmental fluctuations can trigger multiple epimuta- change [2]. The involvement of environmental influences tions in the same individual. This renders fast ecological in generating heritable biological variation is still debated adaptation affecting (complex) adaptive traits more plausible [13, 22], as is the necessity of extending our modern [50]. Hence, recombination is not necessarily a prerequisite evolutionary synthesis [31]. Accumulating evidence indicates for adaptive change, if the latter is driven from the epigenetic that modifications of epigenetic signals are correlated with level. In addition, epigenetic mechanisms may partly defy phenotypic variation within and among species [25, 32– well-understood population processes, such as allelic drift 34], placing epigenetic differentiation even in a macroevolu- (due to potential maintenance of relatively constant epiallelic tionary context. Latest developments regarding the potential frequencies through environmental influence). Being more role of phenotypic plasticity in driving diversification and flexible and dynamic than DNA sequence information, speciation have been discussed elsewhere (e.g., [13, 35]). We variation in epigenetic signals could therefore act as major are hereafter focusing on the impact of heritable epigenetic driving force in rapid adaptive processes. variation on the process of evolution and propose a research Epigenetic variation can have extensive consequences, plan to address its evolutionary significance. even in the absence of genetic variability [45, 50, 51]. Epigenetics may introduce, or reinforce in a back-coupling process with environmental stimuli, major changes that lead 2. Potential Impact of Heritable Epigenetic to strong phenotypic differentiation [52] until becoming VariationonEvolution a real reproductive barrier. Most phenotypic differences between species are genetically controlled, but epigenetic Empirical studies have demonstrated high levels of epigenetic inheritance can be of particular importance for the initial variation within natural populations [25, 36–41]. While development of phenotypic divergence [25]. If adaptive experiments have shown that environmental conditions can and maintained long enough, phenotypic discontinuities override epigenetic signals (e.g., [26, 42, 43]) and increase can become genetically locked and trigger species diver- this variation, few recent studies indicate that natural selec- gence [53]. Modelling studies suggest that epigenetic vari- tion can act directly or indirectly on epigenetic variation [25, ation can promote population divergence by facilitating 38, 39, 44], potentially leading to evolutionary divergence adaptive peak shifts, reducing genetic barriers represented and adaptation. Altogether, epigenetic information provides by fitness valleys in the adaptive landscape [47]. There- an additional source of natural variation, which may be fore, epigenetic novelties have been one of the mecha- particularly important for survival of small populations nisms put forward for saltational speciation [29, 54], but Genetics Research International 3

Type 2

Type 1

acb a b c

Type 2a Type 2a Type 2b Type 1a

Type 2c Type 2b Type 1a Type 1b Type 1b Type 2c Type 1c Type 1c Figure 1: Putative relationships between populations of closely related alternative types (here exemplified with altitudinal differentiation), which lack apparent genome-wide divergence. Below the reflection of the relationships in hypothetical phylogenies is given. Left, single origin of each type, followed by dispersal to other geographical areas. Right, recurrent evolution of the types in several geographic regions under environmental influence. empirical data is not yet available to support or reject such If main discontinuities in phenotypic variation separate a hypothesis. populations along type boundaries (e.g., by morphology or habitat preference), the uniformity within each group and ff 3. A Research Idea constant di erence between the groups might suggest a single origin of each type and subsequent dispersal (Figure 1). Recently developed tools, in combination with traditional However, this seems rather unlikely in absence of genome- methods, can shed light on the complex interactions between wide genetic divergence among the types. An alternative genotype, epigenotype, and environment, and test for their scenario could invoke repeated migration and iterative in individual contribution to phenotypic divergence and evolu- situ formation of each type in alternative environments, with tion. Evolutionary biologists could address the evolutionary very strong and almost identical selection pressures acting relevance of heritable epigenetic polymorphisms by targeting upon different populations of each of the types. closely related ecotypes or species (hereafter types) that ff show phenotypic di erentiation without apparent genome- 3.2. Genetic and Epigenetic Differentiation. Singular versus wide genetic divergence. Such types could be identified, for multiple origin of each type should be tested by inves- example, within asexual lineages or descendants of recent tigating the extent and structure of genome-wide genetic adaptive radiation events. We suggest a multifaceted research and epigenetic divergence within and among populations of plan using an array of molecular techniques and field both types. If populations cluster genetically in disagreement experiments to investigate whether epigenetics is involved in to the type (possibly determined by other factors, e.g., by speciation by triggering phenotypic diversification. geographic proximity), it may be hypothesized that their differentiation is underlaid by epigenetic mechanisms and 3.1. Phenotypic Differentiation. As speciation is facilitated by that types have evolved several times in parallel. Alternatively, the process of divergence, the first question to be addressed local high rates of gene flow combined with strong selection should be whether phenotypic variation in the study group at a few adaptive genetic loci could hypothetically produce is discrete or continuous. Phenotypic variation is a common a similar pattern of highly porous genomes [55]. In such a feature of populations and species, and only a disconti- case, a small number of adaptive (outlier) genetic loci of large nuity in this variation may indicate incipient divergence effect should be responsible for the observed phenotypic and the onset of isolating mechanisms. Therefore, various differentiation. Outlier analyses [56–58] of genetic profiles morphological, anatomical, and physiological traits among provided, for example, by DNA fingerprinting techniques populations of different types should be compared to test such as RAD (restriction site associated DNA) sequencing whether the types form well defined, distinct groups or [59], microsatellites, or AFLP (amplified fragment length whether the extreme phenotypes are linked by individuals polymorphism [60]), could help identifying these loci or with intermediate traits or combinations of characters. In closely linked genomic regions. Positive selection will shape addition, measurements and observations of environmental at target loci a significantly higher differentiation between characteristics (e.g., microclimate, geology, soil, biological populations of the alternative types than the genome-wide interactions) could identify limiting environmental factors, bulk of loci, while loci under purifying selection will show and relate them to anatomical, morphological, and physio- much lower differentiation [61]. On the other hand, if logical specializations. individuals of each group share type-specific epigenetic 4 Genetics Research International patterns and/or mRNA transcripts, differentiation could be other hand, in the case of heritable phenotypic divergence mediated either by overall differences in the epigenome or by that is fully stable even in the alternative environment, a few epialleles. epigenetically triggered adaptation may have been already As epigenetic variation is not detectable in genomic assimilated in the genetic code. surveys of sequence variation, dedicated investigations have Reciprocal transplant experiments together with at- to be employed to address it. In recent years, a variety tempts to grow the different types under the same envi- of genome-wide approaches, including techniques involving ronment across several generations (i.e., between three and next-generation sequencing, have been developed to com- five as a minimum requirement) should be installed to paratively profile epigenetic patterns in nonmodel organisms determine the extent of phenotypic plasticity, and the ability [62, 63]. Cost-effective comprehensive methods include, for of the different types to cope with altered environmental example, fractioning the DNA using C0t filtration [64, 65]to conditions. Growing individuals of the alternative types enrich low-copy regions (mostly genes and their promoters) in a uniform environment across several generations may and sequence this genomic subsample by employing next reveal the heritability of morphological and ecological generation methods and bisulfite sequencing. The latter is characteristics within each of the types (“nature versus nur- a process that converts unmethylated cytosines to uracils, ture”) [75]. Comparatively investigating relevant (epi)loci in which will then appear as thymines after sequencing [66]. transplanted individuals versus controls will pinpoint those Third-generation DNA sequencers, like the recently released patterns that are immediately disrupted by the environment, single molecule real-time (SMRT) DNA sequencer could be and those that persist or, alternatively, are not under the employed for direct detection of DNA methylation [67]and influence of the relevant limiting environmental differences. thus enable much more profound study of both model and Integrating this information and comparing morphological, nonmodel epigenomes. Alternatively, genome-wide DNA anatomical and physiological traits supplemented by a set methylation could be studied using isoschizomers [68, 69]. of fitness components among transplants and controls Similarly as for genetic dataset(s), the epigenetic information will define the links between genotype, epigenotype and couldbesearchedforgeneralpatternsofdifferentiation and phenotype, together with providing additional information for signatures of selection on individual (epi)loci [25, 44]. on the patterns of selection and their targets. This should clarify if ecological and/or morphological diver- According to the mechanisms underlying the observed gence is dependent on just a few loci controlling traits for differentiation, at least two possible outcomes can be antic- local adaptation, or if it is triggered by extensive differences. ipated. If the morphological and/or ecophysiological differ- As the alternative types thrive in different environments, ences are triggered by continuous but nonheritable responses the selective pressures and their magnitude may vary across to local environments (i.e., as a reaction norm [76]), populations. Epigenetic signals will most often suffer from there should be no phenotypic differences between the imperfect heritability; therefore, stronger selection will be progeny of the two types when reared and grown under needed to produce patterns that will be detected as outliers the same conditions. Such a scenario will not (yet) be by statistical approaches. relevant for speciation. On the other hand, if heritable To infer broad, genome-wide regulatory variation, in- epigenetic differences are involved, phenotypic divergence depth quantitative gene expression analyses using next- between individuals of the types should at least partly be generation sequencing (RNA-seq, [70–72]) could be per- retained in a common environment. In the latter case the formed searching for loci with significant expression differ- morphology, anatomy, and physiological properties of the ences between individuals of different types after growing transplanted individuals should reflect their origin rather them under uniform conditions to reduce the momentary- than their current environment. This may go to the extreme dependent noise in rates of expression. In addition, targeting that individuals are maladapted and do not survive under posttranscriptional regulation, small RNA profiles could be alien environmental conditions. compared using an smRNA-seq approach [63, 73, 74]. The The result of these experiments could simultaneously different data types can finally be integrated in functional allow for inferring evolutionary and population dynamics analyses (i.e., gene annotations) to identify correlated com- within the study group. If individuals of alternative types ponents that are part of the same regulatory network. can adapt phenotypically to the habitat of the other and develop the habitat-specific syndromes following transplant 3.3. Heritability of Phenotypic Plasticity and Habitat Speci- experiments, the possibility of frequent gene flow between ficity. If the molecular basis of phenotypic differentiation populations of both types should be considered. This and/or adaptation to divergent environments is identified might as well explain the lack of overall differentiation, within epigenetic rather than DNA sequence divergence, as it prevents lineage sorting and hampers or slows down the next research step would be to investigate how stable speciation. On the contrary, low fitness (i.e., poor per- the phenotypic divergence is. This will also help to assess formance and high mortality) of individuals in the native the stage of speciation in which the group is at present. habitat of the alternative type may point to a differentia- While facilitating population divergence and speciation [35], tion that is strong enough to prevent gene flow between nonheritable phenotypic plasticity will trigger speciation populations. In this case, we may be observing a process only if the environmental conditions are stably different of ongoing speciation, where differentiation starts at the in the alternative localities [35] and gene flow is either epigenetic level, triggering profound changes leading to infrequent or strongly opposed by natural selection. On the segregation in terms of habitat, phenology, and/or biological Genetics Research International 5

Figure 2: Low-elevation Heliosperma veselskyi and high-elevation H. pusillum are diﬀerentiated morphologically and ecologically. Partic- ularly conspicuous is the dense indumentum of sticky glandular hairs on H. veselskyi in comparison to the glabrous leaves and stems of H. pusillum (Photographs: M. Sonnleitner).

interactions. Divergent selection may reinforce this environ- below the timberline. The higher elevation group, including mentally induced specialization/niche segregation and bring H. albanicum, H. pudibundum, and H. pusillum s.str., differs about reproductive isolation. This will eventually result in from the lower elevation group by narrower, glabrous or virtual isolation of gene pools, and ultimately give way to sparsely hairy leaves and often unicellular glands as well stronger overall differentiation by accumulation of genetic as longer seed papillae [78, 81]. By contrast, plants of differences due to the stochastic effects of drift. lower elevations share a denser indumentum with long multicellular glandular hairs and are often sticky (Figure 2). 4. Heliosperma pusillum Group: An Example of Generally, morphological variation is much higher in the lower elevation group, which contains several narrowly an Appropriate Study System distributed taxa [78, 82]. Most of them are endemics of Heliosperma pusillum and allied taxa from the carnation the Balkan Peninsula; only H. veselskyi is restricted to the family (Caryophyllaceae) contain a variety of morphologi- southeastern Alps. The origin and evolution of the lower and cally different taxa (Figure 2) with distinct ecology, which higher elevation groups and the relationships between them are altitudinally or geographically isolated, but genetically are still poorly understood. Recent molecular phylogenetic intermixed (Figure 3) and do not represent independent studies [78] (see also Figure 3) indicate that neither higher evolutionary lineages [78]. Molecular phylogenetic studies nor lower elevation groups are actually monophyletic, but based on AFLPs [77]andsequencesofseveralnuclearand rather inextricably intermingled with each other, indicating chloroplast regions [77–79], show that genetic divergence that one of the groups evolved multiple times from the other. within the group is generally shallow, many taxa seem to Mechanisms involved in the phenotypic diversification of be polyphyletic, and geographically allied taxa often share the two groups, the morphological convergence within each the same genetic constitution. We hypothesize that they group, and the stability of this phenotypic divergence remain either (i) represent fixed ecotypes, that is, differ subtly in unknown, but preliminary evidence suggests that morpho- their DNA coding regions with major phenotypic effects, or logical features remain constant in a common garden, at least (ii) result from middle- to short-term adaptive (epigenetic) in the first generation. The H. pusillum complex is suitable processes, perhaps under the influence of the environment for (epi) genomic and transcriptomic analyses, because all = and independent of actual changes in DNA sequence. All taxa have a relatively small genome (1C 1.32 pg [83]) and so n = x = of them are perennial caespitose herbs that inhabit rocky far no polyploid cytotypes have been found (2 2 24). habitats and shallow caves in mountain ranges of southern In addition, they can be easily grown from seeds and have Europe [78, 80], mostly on calcareous substrates. short generation times, which make them optimally suitable Different authors [78, 81] have subdivided this complex for common garden and transplantation studies. into two ecologically and morphologically distinct groups of taxa: a higher elevation group occurring in damp, open 5. Synthesis and Outlook habitats and among rocks above the timberline and a lower elevation group inhabiting canyons and gorges as Although the possibility of epigenetic inheritance has now well as shallow caves and cliff overhangs with rather dry been established [7, 18, 27, 30, 84] and we are increasingly soils, high atmospheric moisture and poor light conditions understanding the full extent of its role in producing 6 Genetics Research International

epigenotype interact to produce a broad array of short- and long-term heritable combinations. The recently available possibility to profile the epigenome and transcriptome of nonmodel organisms in a high- throughput manner [62, 63, 85] enables thorough investiga- tion of some of the most challenging hypotheses in a modern evolutionary framework, such as achieving and maintaining stable divergence through epigenetic differences. The acquired knowledge also impacts several related domains, from conservation to theoretical evolutionary biology. Inves- tigating recent adaptive radiations with epigenetic markers may be particularly informative. Most traits of ecological significance tend to be continuous or quantitative and appear to be governed by many genes, each of little effect, but with cumulative power [86], resulting in a complex picture of factors and mechanisms acting upon the phenotype. Using appropriate study systems it is now possible to interrogate the links between ecological divergence and many regulatory alterations of small effect or singular major epigenetic switches. In addition, such investigations are expected to pinpoint new loci that are sensitive to epigenetic modifica- tion and unravel information on the rates of spontaneous epimutations in natural populations and their stability over time. Currently accumulating data will offer valuable clues on the establishment of broad regulatory determinants of functional diversity in natural populations. The early evidence we currently hold urges complementing our gene- and genome- centred evolutionary view with a substantial consideration Figure 3: Genetic analyses do not support separation of higher- of epigenetic factors when seeking to understand population altitude Heliosperma pusillum (orange) and lower-altitude H. processes that drive adaptation and divergence [3, 53, 87]. veselskyi (dark blue), but rather indicate an inextricable relationship Using modern technologies, future research will identify the between the two taxa. Unrooted neighbor joining tree based on Nei- exact molecular mechanisms triggering relevant phenotypic Li distances calculated with PAUP from AFLP profiles [77]. divergence and reproductive isolation. We will soon be able to infer the corresponding selection pressures that are responsible for the presence of a particular individual/a particular species in its specific habitat. Understanding how new plant species form and adapt to novel ecological niches is phenotypic variation [19, 25, 39, 40], little research has been crucial to advance our knowledge of evolutionary processes done to systematically study the role of heritable epigenetic active at the population level driving adaptation and spe- variation for speciation. Incorporation of epigenetics into ciation. An increased knowledge of organismic adaptation evolutionary models and empirical studies is only now strategies is also of outstanding importance in the current starting to be attempted (e.g., [28, 49]); however, more context of widespread environmental challenges. It may be empirical information from natural populations is needed a key for predicting effects of climate change and managing for accurate modelling of epigenetic dynamics. Indeed, biodiversity in a sustainable manner. the prevalence of alternative stable epialleles in natural populations, and their significance to phenotypic divergence, ecological interactions and selection in real-world contexts Acknowledgments remain too little explored [3, 41, 53]. The limited relevant data available indicate a stochastic nature of epigenetic varia- The authors are grateful to Brigitta Erschbamer, Karl Hulber,¨ tion, which is continuously being shaped by the influence of Gilbert Neuner, Dieter Reich, Christina Richards and two the environment, and further tuned through natural selec- anonymous reviewers for insightful comments. They thank tion [25, 38, 39]. Therefore, the epigenetic aspect of natural Michaela Sonnleitner for providing skillful photographs. variation may contribute to evolution in a fashion similar Financial support from the “Verein zur Forderung¨ der wis- to genetics, but much more rapidly. Implying heritability senschaftlichen Ausbildung und Tatigkeit¨ von Sudtirolern¨ an of adaptive (i.e., selected) traits, epigenetic inheritance is der Landesuniversitat¨ Innsbruck” for R. Flatscher and from not a contradiction of the Darwinian evolutionary synthesis the Austrian Science Foundation (FWF; project no. P22260- [31], but rather a complex augmentation of the classic B16), and from the European Commission (PERG07-GA- view on genetic inheritance, particularly as genotype and 2010-268462) for O. Paun is acknowledged. Genetics Research International 7

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